In one aspect, the present invention relates to methods for screening for compounds that modulate mitochondrial function by affecting mitochondrial redox potential. Methods of the invention also can be used to test for mitochondrial fitness.
Nearly every cell of the body contains organelles called mitochondria which produce most of the energy used by the body. Certain cells with high metabolic rates, such as heart muscle cells, may contain thousands of mitochondria.
Energy derived from the utilization of substrates is required in order to maintain the non-equilibrium state necessary to carry out the basic functions of the cell (e.g. contraction, secretion, electrical propagation, ion pumping, cell division). There are three major steps involved in cellular energy production. First, large food molecules are broken down into smaller units. Polysaccharides are converted to simple sugars, fat is converted to fatty acids and proteins are converted to amino acids. Second, multiple pathways convert these three different molecules into a common precursor, acetyl CoA, for further oxidation by the mitochondria. Third, the Krebs cycle (a.k.a. the citric acid or tricarboxylic acid cycle) and oxidative phosphorylation convert acetyl CoA into ATP, the energy storage molecule directly utilized by the energy consuming reactions in the cell.
Mitochondrial substrate oxidation is a multi-step process that includes a series of reactions which transfer electrons from the initial substrate to nicotinamide adenine dinucleotide (NAD+) to produce NADH which is then reoxidized by passing electrons through the electron transport chain to the ultimate electron acceptor oxygen. In the process of electron transfer, protons (H+) are pumped out of the mitochondrial matrix (an intracellular aqueous compartment bounded by the inner and outer mitochondrial membranes between it and the cell""s cytoplasm) across the mitochondrial inner membrane (IMM) resulting in the establishment of a proton gradient. This proton concentration gradient, together with the large membrane voltage generated by the active charge movement across the IMM, provide the driving force for proton movement back into the matrix (the protonmotive force) which will be utilized by a specific IMM protein (the mitochondrial ATP synthase) to convert ADP (adenosine diphosphate) to ATP (adenosine triphosphate). ATP thus produced is transported out of the mitochondria and is available to perform the work required by the cell.
The relative impermeability of the IMM to ion leak and the presence of energy conserving pumps and exchangers in the membrane allows for the efficient utilization of the protonmotive force for cellular energy production rather than expending it for reestablishing the IMM gradient. Despite this requirement for maintaining a high resistance membrane, the IMM contains a number of energy dissipating high conductance pathways for ion and/or solute movement. The physiological role of some of these pathways is apparent, for example, the pyruvate transporter is required to import substrate into the matrix for oxidative phosphorylation; however, in other cases, the physiological importance of IMM ion conducting pathways is still unknown. A well known example is the mitochondrial megachannel (MMC) or permeability transition pore (PTP). When activated, this large non-selective channel rapidly de-energizes the mitochondrion and has been implicated in several pathophysiological states. Other known high conductance pathways include the calcium uniport, the mitochondrial inner membrane anion channel, the mitochondrial uncoupling protein of brown fat mitochondria, and the mitochondrial ATP-sensitive potassium channel. A number of electrogenic (e.g., the adenine nucleotide transporter, the glutamate-aspartate transporter, the Naxe2x80x94Ca exchanger), proton-compensated electroneutral (e.g., the glutamate, pyruvate, and malate-citrate transporters) and electroneutral (e.g., the malate-phosphate, malate-ketoglutarate, carnitine, ornithine, and neutral amino acid transporters) are also present in the IMM and may influence the mitochondrial energy state. Although some of these channels and transporters have been well studied in isolated mitochondria, for lack of a useful index of mitochondrial activity in intact cells, much less is known about their regulation or sensitivity to pharmacological agents in intact cells.
The mitochondria are essential for efficiently providing ATP for carrying out the myriad functions of the cell, particularly in tissues with a high energy demand, such as muscle and brain. Consequently, defects in mitochondrial energy metabolism are usually associated with significant functional deficits or death. The pathophysiologies related to mitochondrial dysfunction can be either primary or secondary. In primary mitochondrial diseases, a genetic defect (either inborn or acquired) in a mitochondrial protein may lead to the incorrect assembly or catalytic activity of the protein, thus disrupting or impairing the entire biochemical pathway. Secondary mitochondrial disorders may arise from the accumulation of toxic products within the cell (including oxygen free radicals), the accumulation of inhibitory metabolites, or lack of cofactors required for mitochondrial metabolism.
Mitochondrial cytopathies of differing origin often lead to similar clinical symptoms. Commonly the disorders are first expressed in the most metabolically active tissues. They may present as muscle weakness and fatigue, mild muscle ache, or severe (and sometimes lethal) lactic acidosis during exercise. In many cases, these muscle deficiencies are also associated with central nervous system disorders, referred to as mitochondrial encephalomyopathies (e.g, KSS, Kearnes-Sayre syndrome; MERRF, myoclonus epilepsy with ragged red fibres; MELAS, mitochondrial encephalomyopathy/lactic acidosis/stroke). These disorders may arise from point mutations in or deletions of large segments of mitochondrial DNA. In many cases, the specific enzyme affected is known (e.g. pyruvate dehydrogenase deficiency). Similarly, defective nuclear encoded proteins involved in mitochondrial metabolism or drugs interfering with respiration (e.g., AZT or Adriamycin) can also lead to mitochondrial cytopathies.
Mitochondrial cytopathies can be classified by the site of the defect in mitochondrial oxidation. Defects in substrate transport (e.g. carnitine or carnitine-palmitoyl-transferase deficiencies), substrate metabolism (e.g. deficiencies in pyruvate dehydrogenase, pyruvate carboxylase, fatty acid oxidation, or organic acid metabolism), Krebs cycle activity (e.g. defects in oxoglutarate dehydrogenase or fumarase), the respiratory chain (NADH-Q reductase or cytochrome deficiencies), or energy coupling (ATP synthase defect, mitochondrial uncoupling diseases).
Cumulative alterations in mitochondrial metabolism have been suggested as an underlying cause of diseases associated with aging, including Alzheimer""s and diabetes mellitus. Furthermore, mitochondria are the site of initiation of programmed cell death (apoptosis) and probably are the key factor in determining whether or not a cell will recover from an ischemic insult or proceed to necrosis.
Thus, mitochondria are central to the survival and function of the cell under normal conditions and play a major role in adapting to environmental stress.
Thus, it would be desirable to have a method of studying mitochondrial function as well as methods of assessing the effect of different chemicals on mitochondrial function. It would be particularly desirable to identify compounds that selectively modulate mitochondrial function. It would be also useful to detect compounds that affect mitochondrial KATP channels.
The present invention relates to methods of assaying for mitochondrial function and more particularly to methods of identifying compounds that selectively modulate mitochondrial function. In one aspect, the present invention relates to methods of detecting compounds that can positively impact mitochondrial function and increase cell energy output. In a related aspect, the invention relates to methods of detecting compounds that can decrease mitochondrial function in diseased cells. The present invention has a variety of useful applications including use in screens to detect compounds that can enhance overall health and fitness.
More particularly, the invention includes methods for detecting the effects of agents acting on mitochondrial metabolism in intact cells by utilizing endogenous redox potential sensitive fluorophores located in the mitochondria. The methods of the invention can enable low cost high throughput screening of compounds which modify the functional state of mitochondria for therapeutic applications. Methods of the invention are applicable to the discovery of agents which modify the activity of any of the steps in energy metabolism. For instance, an exemplary and preferred application detects mitochondrially active agents in intact cardiac cells.
In general, the methods of the present invention are useful for detecting drugs that alter mitochondrial function. For example, in one aspect, the invention provides a drug detection assay by measuring endogenous fluorescence in intact cells. In a normal oxygenated medium, the mitochondrial matrix is significantly reduced. Drugs that decrease the membrane potential across the inner mitochondrial membrane cause oxidation of the matrix, which is detected as a change in endogenous fluorescence by the methods of the present invention. Accordingly, the methods of the present invention are well-suited to detect compounds that can selectively enhance or decrease mitochondrial function.
In the present methods, cells are cultured and illuminated at wavelengths suitable to excite endogenous fluorescence. Preferably the fluorescence is due to changes in the redox state of endogenous molecules located in the mitochondria. These endogenous molecules function as reporters of mitochondrial oxidation state. Preferred molecules include endogenous proteins that comprise fluorescent molecules such as a flavin moiety, or endogenous fluorescent molecules such as NAD.
One aspect of the invention relates to a method for identifying a compound capable of modulating mitochondrial function comprising contacting a eukaryotic cell with one or more candidate compounds and detecting a change in the mitochondrial redox state. Preferably, endogenous fluorescence of the cell mitochondria is indicative of a change of redox state. The change in the redox state is an increase or decrease in the state of the mitochondria oxidation. That change is typically related to a suitable control assay as described below.
In certain preferred methods of the present invention, the fluorescence is measured of a nicotinamide adenine dinucleotide (NAD) or a flavin adenine dinucleotide (FAD) moiety, such as a protein comprising a linked flavin adenine dinucleotide (FAD) moiety. In embodiments comprising such a FAD-linked protein, preferably the FAD-linked protein is linked to a protein component of a mitochondrial redox pathway.
In preferred methods, detection of the mitochondrial redox state further comprises measuring a change in fluorescence of an NAD molecule or FAD-linked enzyme, and correlating that change to a control assay comprising a mitochondrial oxidizing or reducing agent. Illustrative oxidizing agents include dinitrophenol, and illustrative reducing agents include cyanide.
In certain embodiments of the present invention, the cell is contacted with a plurality of candidate compounds or a library of candidate compounds.
In certain embodiments the steps of contacting a eukaryotic cell with one or more compounds and detecting the change in the mitochondrial redox state of the cell are performed a number of times substantially simultaneously. These steps can be performed e.g. in a multi-well plate.
The eukaryotic cell used in certain methods of the present invention comprises a cardiac cell or a precursor cell thereof. In certain preferred embodiments, the eukaryotic cell is a cardiac cell or precursor cell thereof that is immortalized. In other preferred methods, the cardiac cell or precursor cell thereof is a primary cell. The cell may comprise a ventricular myocyte or a skeletal myoblast.
The present invention relates to methods for detecting many different types of drugs capable of modulating mitochondrial function. The present invention also relates to methods wherein the candidate compound activates a mitochbndrial KATP channel. Further, it relates to methods of assaying the activity of mitoKATP channels using fluorescence methods. In certain preferred embodiments, the compound does not substantially activate a sarcolemmal KATP channel.
In certain embodiments of the present methods, the cell is contacted with the candidate compound(s) in vitro. In other embodiments, the cell is contacted with the candidate compound(s) in vivo. In yet other embodiments the cell is a tissue and the tissue is treated with the candidate compound ex vivo.
The present invention further relates to a method for detecting a compound capable of modulating mitochondrial redox potential, the method comprising:
a) providing a population of eukaryotic cells;
b) contacting a first portion of the cells with one or more candidate compounds;
c) contacting a second portion of the cells with a known mitochondrial oxidizing or reducing agent; and
d) measuring a difference between mitochondrial fluorescence produced in steps b) and c).
It will be appreciated that in cases where the mitochondrial fluoresecence of a known oxidizing or reducing agent is known, it will not always be necessary to perform step c), above.
In particular embodiments of this method, the cells are ventricular cells and the method further comprises measuring mitochondrial KATP ion channel currents in those cells. In some embodiments of this method, the compound activates a mitochondrial KATP ion channel in the cells. In other embodiments, the method further comprises measuring sarcolemmal KATP ion channel currents in the cells. In some examples of such methods, the drug does not substantially activate the sarcolemmal KATP ion channel currents at comparable concentrations.
In certain embodiments of the methods, the mitochondrial fluorescence is activated by a light at a wavelength of from about 250 to about 650 nm. In these embodiments, the step of detecting or measuring is accomplished by fluorescence microscopy.
The methods of the present invention are applicable to nearly any eukaryotic cell. Preferred cells comprise detectable mitochondrial fluorescence. For example, such cells will often include cells from highly energetic tissues such as muscle and particularly cardiac and skeletal muscle cells. In addition certain rapidly dividing cells can also be used such as cancer cells (primary or cultured cell line) and immature cells (e.g., hemapoeitic cells). However, in preferred embodiments, the eukaryotic cells are selected from the group consisting of H9C2 (rat ventricular myocyte-derived cell line), AT-1, HL-1 (atrial tumor derived cell line) and C212 (murine skeletal muscle-derived cell line) cells.
Preferably, the methods identify a candidate compound drug that modulates mitochondrial oxidation (e.g. mitochondrial flavoprotein oxidation) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, in a mitochondrial redox assay of the invention relative to a control (i.e. the same assay where the candidate compound has not been exposed to the test cells). The EC50 of identified candidate compounds is preferably no more than about 10 xcexcM in a standard whole-cell-patch-clamp assay.
The invention further relates to a method of detecting the activity of a mitochondrial ion channel or mitochondrial transporter comprising contacting a eukaryotic cell with one or more candidate compounds and detecting a change in the mitochondrial redox state as indicative of the activity of the ion channel or transporter.
The invention also further relates to drug compounds obtained by the above-described method.